The invention relates to the field of microscopy, and particularly related to the field of coherent anti-stokes Raman scattering microscopy.
Coherent anti-stokes Raman scattering (CARS) microscopy provides for the imaging of chemical and biological samples by using molecular vibrations as a contrast mechanism. In particular, CARS microscopy typically uses two laser fields, a pump electromagnetic field with a center frequency at ωp and a Stokes electromagnetic field with a center frequency at ωs. The pump and stokes fields interact with a sample and generate a coherent anti-Stokes field having a frequency of ωAS=2ωp−ωs in the phase matched direction. When the Raman shift of ωp−ωs is tuned to be resonant at a given vibrational mode, an enhanced CARS signal is observed at the anti-Stokes frequency ωAS.
Unlike fluorescence microscopy, CARS microscopy does not require the use of fluorophores (which may undergo photobleaching), since the imaging relies on the vibrational contrast of biological and chemical materials. Further, the coherent nature of CARS microscopy offers significantly higher sensitivity than spontaneous Raman microscopy. This permits the use of lower average excitation powers (which is tolerable for biological samples). The fact that ωAS>ωp, ωs allows the signal to be detected in the presence of one-photon background fluorescence. CARS microscopy provides information about the intrinsic vibrational resonances of a sample with high sensitivity, allowing for label-free, chemically-specific imaging.
For example, U.S. Pat. No. 4,405,237 discloses a coherent anti-Stokes Raman spectroscopic imaging device in which two laser pulse trains of different wavelengths, temporally and spatially overlapped, are used to simultaneously illuminate a sample. The '237 patent discloses a non-collinear geometry of the two laser beams and a detection of the signal beam in the phase matching direction with a two-dimensional detector.
U.S. Pat. No. 6,108,081 discloses a different method and apparatus for microscopic vibrational imaging using coherent anti-Stokes Raman scattering. In the apparatus of the '081 patent, collinear pump and Stokes beams were focused by a high numerical aperture (NA) objective lens. The nonlinear dependence of the signal on the excitation intensity ensures a small probe volume of the foci, allowing three-dimensional sectioning across a thick sample. The signal beam is detected in the forward direction.
There is also a nonresonant contribution to the CARS signal, however, that does not carry chemically-specific information that can distort and even overwhelm the resonant signal of interest. This nonresonant contribution provides background with no vibrational contrast from which the desired signal must be filtered or somehow distinguished. For example, a conventional lateral CARS intensity profile of a 535 nm polystyrene bead embedded in water includes a substantial amount of CARS background from water in addition to the characteristic CARS signal from the bead. The presence of this background from the isotropic bulk water has hindered efforts to increase the sensitivity of CARS imaging, particularly in biological applications. The CARS background is caused by electronic contributions to the third order nonlinear susceptibility. There exists a non-resonant contribution to the CARS signal of the sample of interest as well as of the surrounding isotropic bulk medium (i.e., solvent), which is independent of the Raman shift, ωp–ωS.
One approach to reducing the non-resonant background field in CARS spectroscopy is to take advantage of the fact that the non-resonant background has different polarization properties than the resonant signal. For example, see Polarization-Sensitive Coherent Anti-Stokes Raman Spectroscopy, by Oudar, Smith and Shen, Applied Physics Letters, June 1979, pp. 758–760 (1979); and Coherent ellipsometry of Raman Scattering of Light, by Akhmanov, Bunkin, Ivanov and Koroteev, JETP Letters, Vol. 25, pp. 416–420 (1977), which employ non-collinear excitation beams with different polarization directions.
U.S. Pat. No. 6,798,507 discloses a system in which the pump and Stokes beams are polarized, and a polarization sensitive detector is employed. In high resolution CARS microscopy, however, tightly focused collinear excitation beams are sometimes necessary. It is known that tightly focusing polarized beams will result in polarization scrambling. See Principles of Optics, Born and Wolf, Pergaman Press, 1989, pp. 435–449.
U.S. Pat. No. 6,809,814 discloses a system in which a CARS signal is received in the reverse direction (epi-direction) from the sample. The epi directed signal, however, is significantly smaller than the forward directed signal, and a stronger signal may be desired for certain applications.
There is a need, therefore, for a system and method for providing improved sensitivity of CAS microscopy for certain applications, and in particular, to provide a CARS detection scheme that reduces the non-resonant background hence yields a higher signal-to-background ratio.